II. A. Importance of glycosylation in GRP-R mediated cell modulation. Previous studies by us show using enzymatic deglycosylation that glycosylation of the gastrin-releasing peptide receptor but not the neuromedin B receptor [NMB-R] alters receptor affinity. To determine the number of consensus sequences which glycosylation sequence effects receptor affinity as well as whether glycosylation also is important for the receptor processes we used site-directed mutagenesis of the GRP-R, all of the potential N-linked glycosylation sequences in the GRP-R were altered either singly or together (N5Q, N20G, N24Q, N191Q). We found using cross-linking studies that all 4 sites were glycosylated [13kD at Asn5, 10kD at Asn24, 5Kd at, Asn24, 9kD at Asn191]. Asn24 and Asn191 were important for cell surface expression of the GRP-R. Elimination of Asn191 was responsible for the decreased affinity of the 80% deglycosylated receptor because the N5, 20, 24Q mituant had normal receptor affinity and coupling. Elimination of Asn5, Asn20, and Asn24 altered receptor down-regulation and desensitization but not receptor internalization. GRP-R glycosylation is important for cell surface expression, G-protein coupling, densitization and down regulation. II. B. Signal Transduction of the human PACAP receptor splice variants. In the present study the human PACAP receptor gene was cloned. Alternative splicing about two exons of the gene allowed for four major splice variants. Each splice variant cDNAs was stably expressed in NIH/3T3 cells at similar receptor densities. PACAP had similar affinity and potency for stimulating either adenylate cyclase or PLC for each splice variant. However, each splice variant differed in their ligand-stimulated maximal response. Therefore, unlike the rat, PACAP binds and stimulates signal transduction with nearly equal affinity and potency for each of the receptor splice variants although with varying efficacy for the stimulation of PLC. These results suggest a novel and potentially important mechanism for a single hormone to not only coupled to dual signal transduction cascades but also elicit tissue-specific transduction cascades but also elicit tissue-specific differential activation of PLC in humans. III. C. Molecular basis for CCKB receptor gastrin selectivity. The two known receptors mediating the actions of cholecystokinin (CCK) and gastrin. CCK type A (CCKAR) and CCK type B (CCKBR) receptors, are G protein-coupled receptors. To determine the structural basis for the selectivity of the CCKBR for gastrin, a series of CCKB/AR chimeras were constructed in which segments of the CCKBR were replaced with the corresponding segments of the CCKAR. The sequence spanning from transmembrane domain III (TM III) to TM V was the only segment that resulted in the selective loss of gastrin affinity. Using site- directed mutagenesis in this segment of the CCKBR, a sequence of 5 amino acids in the second extracellular loop was found to be responsible for this 100-fold selective loss in gastrin affinity. These results present for the first time the identification of the amino acid sequence of the CCKBR conferring the majority of the selectivity for gastrin.